Ahmad et al. BMC Res Notes (2016) 9:477 DOI 10.1186/s13104-016-2271-4 BMC Research Notes

RESEARCH ARTICLE Open Access Genetic diversity of the Tibetan (Pantholops hodgsonii) population of , , its relationship with other populations and conservation implications Khursheed Ahmad1, Ved P. Kumar2, Bheem Dutt Joshi2, Mohamed Raza1, Parag Nigam3, Anzara Anjum Khan1 and Surendra P. Goyal2*

Abstract Background: The (Pantholops hodgsonii), or chiru, is an endangered antelope, distributed in [, Xizang, , Zhuolaihu Lake (Breeding habitat)], and India ( and Ladakh). There is a global demand for the species prized wool, which is used in weaving shahtoosh . Over the years, the population of the Tibetan antelope has drastically declined from more than a million to a few thousand individuals, mainly due to . Field studies undertaken in Ladakh, India also indicated winter migration of the population to . Migra- tion between winter and calving habitats is well established to be female-biased across the Qinghai (QTP). For effective conservation planning, genetic characterization is considered the best way to understand the likely impact of threats for ensuring the long-term viability of the population. In this regard, genetic characteristics of all Chinese populations are well-studied using mitochondrial and microsatellite markers, but information is lacking for the Indian population. Therefore, using the control region marker, we document for the first time the genetic variation of the Indian population of the Tibetan antelope, the extent of migration and its relationships with other populations of China. Results: The partial fragment of control region (259 bp) marker was successfully amplified in 30 Tibetan antelope samples that were collected from the Chang Chenmo Valley in eastern Ladakh, India. We also retrieved control region sequences (n 88) available in the public domain from GenBank of different Chinese populations. Low levels of nucleotide (π; =0.004) and haplotype (hd; 0.543) diversity were observed in the Indian population when compared to Chinese populations (π 0.01357–0.02048 and hd 0.889–0.986). Commonly used indices (Tajima’s D and Fu’s Fs) were analyzed for inferring= the demographic history= of the Indian populations, and all values were negative indicating population expansion or demographic equilibrium, though nonsignificant. We observed five haplotypes in the Indian population, and these were not reported in previously studied populations of QTP. Bayesian-based phylogenetic analysis indicates the presence of four clades, however, the posterior probability support for three of these clades is weak (<0.5). Of these, the Indian population formed a distinct clade, whereas the Chinese populations exhibited shared haplotypes, and no geographic structure was observed. Median-joining network analysis was conducted for 46 haplotypes in the overall population, except the samples from India which showed a star-like topology. The Indian population is separated by one median vector from the Chinese population. Conclusions: The present study revealed the presence of different sub-clades in the Bayesian phylogenetic tree and five new haplotypes only in the Indian population or sampling location. Furthermore, in the phylogenetic tree,

*Correspondence: [email protected] 2 Wildlife Forensic and Conservation Genetics Cell, Wildlife Institute of India, Post Box #18, Chandrabani, Dehradun, Uttarakhand 248001, India Full list of author information is available at the end of the article

© The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ahmad et al. BMC Res Notes (2016) 9:477 Page 2 of 10

Indian haplotypes of Tibetan were clustered with the haplotype reported in the Chinese population of the Xinjiang region. Median-joining network analysis showed shared haplotypes pattern in all populations of QTP except the samples from India which showed new haplotypes. Given the presence of low nucleotide and haplotype diversity in eastern Ladakh populations and limited information available for populations of the western side in its range, we suggest to include genetic studies of Tibetan antelope populations around Aksai Chin (Fig. 1) under the proposed transboundary agenda between India and China and assess relationships with other populations. Such understand- ing would enable the planning of conservation strategies for ensuring long-term survival of westernmost populations in its range, and if required, it would establish connectivity with the other populations. Keywords: mtDNA, Shahtoosh wool, Poaching and conservation, Tibetan antelope

Background The Tibetan antelope is endemic to the Qinghai The Tibetan antelope (Pantholops hodgsonii), also known Tibetan Plateau (QTP) and occupies open-high eleva- as chiru has very recently been reclassified on the Inter- tion alpine and desert steppe habitats in the mountainous national Union for Conservation of Nature (IUCN) Red terrain with frequent occurrence at elevations between list as near threatened due to the recovery of some popu- 3250 and 5500 m [3–6]. The present population occurs, lations [1]. The species is listed in Appendix 1 of the Con- almost exclusively, in about 800,000 km2 of the Chinese vention On International Trade In provinces of Xizang (XZ), Xinjiang (XJ), Qinghai (QH) (CITES) (1979) [2], and in Schedule I of the Indian Wild- [7–11], Zhuolaihu Lake (breeding habitat; BH), and in life (Protection) Act 1972. It is a Class I protected species very small numbers in north-western India (Aksai Chin under the Law of the People’s Republic of China on the and Ladakh), mainly in summer [12–14] (Fig. 1). The Protection of Wildlife (1989). Tibetan antelope has rarely been found in [15–18].

Fig. 1 Current distribution of Tibetan antelope (Pantholops hodgsonii) in the Tibetan Plateau of the Chinese provinces of Tibet, Qinghai, Xinjiang and India Ahmad et al. BMC Res Notes (2016) 9:477 Page 3 of 10

At the beginning of the last century, more than a mil- scarves [29–31]. These garments can be sold for $1000– lion Tibetan antelopes were present, but more than 50 % 5000 USD for a single piece and sometimes as much as of individuals have been reduced in just 20 years of the $15,000–17,600 USD depending on the length [31, 32]. 20th century due to habitat fragmentation and increased It has been documented that when large numbers of ani- human activity such as poaching, hunting and smuggling mals are harvested or removed from the wild population of product [5, 10, 11]. Approximately 75,000 Tibetan due to poaching and other factors, the genetic diversity antelopes are now reported in their distributional range and population structure is severely affected by decreas- [19, 20]. Zhuolaihu Lake is known as one of the major ing effective population size [33, 34]. breeding habitats (BH) or calving ground for Tibetan A limited number of studies have been conducted so far antelopes present in the Qinghai province where dur- on the genetic aspects of Tibetan antelope populations, ing the summer season, thousands of females migrate and these studies have revealed that Tibetan antelopes there from wintering habitats to breed [21]. It has been have female-biased dispersal with a high mutation rate of suggested that the migratory behavior exhibited by the mitochondrial DNA (mtDNA) [10, 19], which is a good Tibetan antelope promotes genetic exchange between marker for identifying female-based linkages between different meta-populations and maintains all the popula- populations [35–38]. Hence, we hypothesized that the tions together as one panmictic population [21]. Tibetan antelope population of India is also genetically In India, Tibetan antelopes are found in Daulat Beg connected with the rest of the populations because of Oldi (DBO) and the Chang Chenmo Valley (CCV) in the observed migratory behavior on QTP. Therefore, north–east Ladakh (Fig. 1) [13, 14]. The average altitude we describe, for the first time, genetic variation in the of these areas is 4725–5500 m, and Tibetan antelopes hypervariable control region of the mtDNA genome and are found between the altitudes of 4709–4964 m (mean describe its relationships with other populations. 4797 ± 27.9 m). These areas are mostly surrounded by glaciers and rivers. Unequal individual numbers were Methods sighted from the DBO: 230 individuals during 2005 and Sample collection 45 individuals in 2006. In the CCV, a total of 55–60 indi- Four hair samples and 26 fecal samples were collected viduals of Tibetan antelopes were reported during 2005. from different locations in Chang Chenmo Valley These two Indian populations (DBO and CCV) are iso- (Ladakh District), Jammu and , India (IND 1–30) lated from one another due to mountains and river barri- (Table 1) during field work. Collected hair samples were ers. In DBO, both males and females have been reported kept in an envelope and supplied with the sampling loca- [22, 23], whereas in CCV, only males have been seen [11]. tion and sample ID. The fecal pellets were collected and Though migration from wintering to calving habitats is air dried to remove all moisture. The pellets were then well-documented on QTP, little is known for such move- kept in sterile vials containing silica gel and brought into ments of the population of India. It has been reported the laboratory. that the male Tibetan antelope population in CCV in India is resident [23]. In contrast to these males, individ- DNA extraction and PCR amplification ual females travel distances of 300–400 km in late spring Genomic DNA was extracted from hair samples using a and early summer [24]. About 300 individuals season- hair extraction kit (Merck, GeNei™), and pellet genomic ally migrate to India from the Chinese provinces of Tibet DNA was isolated using a commercially available kit (Xinjiang area) [12], though it is not clear whether this (Qiagen DNeasy Stool Kit, Qiagen, Germany) according population is truly migratory like the other populations to the manufacturer’s instructions. To avoid contamina- of QTP [21, 25, 26] or is resident [22, 23]. tion, each sample was processed separately using new Tibetan antelopes are poached for their wool, known sterile blade during DNA extraction, and negatives con- as ‘shahtoosh,’ a layer of dense, fine wool on their skin trol were included to monitor contamination. Amplifi- that traps warm air, from which fine yarn is produced. cation of the hypervariable fragment of control region This commands a high price [27, 28]. Shahtoosh is dif- (259 bp) marker was amplified using an ABI 2720 ther- ferent from other wools as it can’t be sheared or combed mocycler with a pair of primers [38]. The polymerase because the fibres are very short and attached to the chain reaction (PCR) was conducted in 20 µl reaction bases of guard hairs; in turn, it can only be obtained volumes that contained 1× PCR buffer, 25 mM MgCl2, after sacrificing the . One individual yields about 10 mM dNTPs, 10 µM of each primer, 0.5 U Taq poly- 125–150 g of shahtoosh, and weaving one requires merase (MBI, Fermentas, USA), and approximate 20 ng wool from four to five individuals [11]. It is estimated of genomic DNA. To check for DNA contamination, that every year, more than 20,000 individuals are poached a negative control was set up with a PCR master mix. for their fine wool to make shahtoosh shawls and The PCR cycling conditions were the following: initial Ahmad et al. BMC Res Notes (2016) 9:477 Page 4 of 10

Table 1 Details of samples and mtDNA diversity (π and hd) of Tibetan antelope using control region (259 bp) marker from five locations Population n Variable site No. of haplotypes Nucleotide diversity (π) Haplotype diversity (hd) Tajima’s D Fu’s Fs Source*

IND 30 9 5 0.00458 0.543 0.71389 0.295 Present study − − XJ 17 15 12 0.01357 0.956 1.29957 5.770 Ruan et al. [10] − − XZ 18 19 11 0.01474 0.889 1.45806 3.542 Ruan et al. [10] − − QH 18 19 9 0.02048 0.895 0.5069 0.376 Ruan et al. [10] − − BH 35 21 17 0.01712 0.921 0.70218 5.533 Zhang et al. [19] − − Overall 118 83 54 0.01810 0.927 1.32871 23.551 − − * The analysis is only performed with the 259 bp denaturation (94 °C) for 5 min, followed by 40 cycles of population sizes through time via MCMC procedures. denaturation (40 s at 94 °C), annealing (50 s at 56 °C), The constant population size coalescent model was extension (40 s at 72 °C), and a final extension at 72 °C the basic assumption used for this approach. Together, (10 min). Amplification of PCR was checked on gel elec- among-site rate heterogeneity across all branches and trophoresis using 2 % agarose gel. a strict molecular clock were used for this calculation. 7 Markov chains were run for 2.5 × 10 generations and DNA sequencing and analysis were sampled every 1000 generations with the first 2500 The PCR products were purified using ExoSAP (exo- samples discarded as burn-in. Other parameters were set nuclease I-shrimp alkaline phosphatase) and finally as default values, and results were visualized in TRACER sequenced on an ABI 3130 genetic analyzer using a Big version 1.6 [44]. Dye Terminator v 3.1 Kit (Applied Biosystem, USA). Results and discussion Sequences of the control region (n = 88) of different Chinese populations of Tibetan antelope were retrieved The partial fragment of the control region (259 bp) was from the work of Ruan et al. [10] and Zhang et al. [19] sequenced successfully for all 30 samples of the Indian (Table 1). The ( hircus) was used as an out- population. Nine polymorphic and two singleton variable group in our study. The alignments of the control region sites were found (Table 1) which defined a total five H. sequences of Tibetan antelopes with the complete con- After combining the control region sequences of the four trol region of goat (AF533441) were aligned using Clustal Chinese populations (n = 88) and the Indian population W as implemented in BioEdit version 7.0.9.0 [39]. The (n = 30), a total of 83 variable (polymorphic) sites were sequence divergence between haplotypes (H) was deter- observed which constitutes a total of 46 H considering mined using a MEGA version 6.0 [40]. We calculated the gaps or missing sites. The nucleotide frequencies were as haplotype (hd) and nucleotide diversity (π) using DnaSP follows: A-41.14 %, T-28.20 %, C-21.17 %, and G-9.49 %. version 3.5.1 [41] to check the level of genetic diversity The overall hd and π within the Indian population were in the Indian population and compared these diversity 0.543 and 0.00458. The combined analysis of the Indian indices with other Chinese populations of Tibetan ante- and Chinese samples revealed that the overall haplotype lope. Neutrality tests were performed using the software and nucleotide diversity were 0.927 and 0.01810, respec- Arlequin version 3.0 [42] and DnaSP, version 3.5.1 [41] tively (Table 1). The values of nucleotide (0.00458) and as these values indicates recent population expansion or haplotype diversity (0.543) indicates that the low diver- contraction. Phylogenetic tree using the Bayesian infer- sity within the Indian population in comparison to the ences (BI) were conducted in BEAST version 2.1.3 [43]. Chinese populations (π = 0.01357–0.02048; hd = 0.889– A median-joining haplotype network analysis was con- 0.956). Additionally, these diversity indices were also structed using the Network version 4.5.1 (http://www. lower when compared with the data set of Ruan et al. fluxusengineering.com). [10] (π 0.02178; hd 0.997) based on more than 1 kb pair Patterns of historical demography were inferred from (Table 1). the estimates of effective population size over time using A comparison of diversity indices to other endangered the Bayesian skyline plot (BSP) implemented in BEAST populations of antelope and species indicate similar version. 2.1.3. We used the substitution rate according to ranges of diversity viz. ( equi- Zhang et al. [19] which was used for Tibetan antelopes nus; π 0.000–0.030; hd 0.500–1.00) [45], (2 % per million year for the control region gene). This (Saiga tatarica; π 0.00478; hd 0.918) [46], and Kashmir method estimates a posterior distribution of effective , ( elaphus hanglu; π 0.008; hd 0.589) [47]. Ahmad et al. BMC Res Notes (2016) 9:477 Page 5 of 10

Observed low genetic diversity in Chiru is similar to the population expansion. Likewise, published studies on Kashmir red deer population which is documented to QTP have also reported negative values in demographic have undergone a major decline due to habitat degra- history indices and a unimodal mismatch distribution dation and anthropogenic pressure [48]. However, the pattern in overall populations except one as reported nucleotide diversity observed in the Indian population of by Du et al. [25]. However, Zhang et al. [19] found the Tibetan antelopes is half of that observed in Kashmir red presence of weak multimodal mismatch patterns; yet, deer (0.008), which is the only surviving population of the shape was in agreement with the model of popula- this species in the Indian subcontinent and has declined tion growth. In addition, we also observed non-signifi- in its distribution range. Hence, red deer population of cant sum of square deviations and raggedness in all the India has been suggested to be promoted in the endan- populations, which indicates that data are a relatively gered category [47]. Observed loss of genetic diversity of good fit to a model of population expansion [49]. All Indian population in comparison to Chinese populations these analyses, including ours, reveal historic popula- of Tibetan antelopes requires immediate conservation tion expansion, and suggests that colonization of the attention. Indian population was part of this expansion. However, The sequence divergence between the different popu- we observed relatively higher values of the raggedness lations of Tibetan antelope was 0.014–0.028 %, whereas index (though non-significant in the Indian population) it is low (0.019) between the Indian and the Xinjiang which may suggest stable or bottlenecked populations, populations in comparison to the other three Chinese whereas these values in populations of QTP were low populations (Table 2). The Indian population indicates [19]. Bayesian skyline plot (BSP) analysis of the Indian high Fst (>0.50; p = 0.001) with other Chinese popu- population (Fig. 2) indicated the population remained lations, whereas values ranged only between 0.00069 constant for a long time, and there has been a recent and 0.0764 in spite of large geographical distances decline ca. around 5 Kyr. Such analysis undertaken for among Chinese populations (305–815 km) (Fig. 1). Our the QTP population has highlighted constant popula- analysis indicates that the Indian population has been tion during early-mid Quaternary and expansion dur- isolated from the rest of the Chinese populations; how- ing mid Holocene. However, declines in population size ever, the level of sequence divergence indicates that the were also observed around 5 Kyr [25]. These declines Chinese population of Xinjiang is closer to the Indian may have been due to anthropogenic activities and envi- population than the rest of the populations. Matel test ronmental changes [10, 19, 25]. analysis indicates isolation by distance (r = 0.6154; The Bayesian phylogenetic tree, including samples p = 0.90) in all the Tibetan antelope populations but from the Chinese populations, indicates the evolution- p value is not significant. We performed Tajima’s D ary relationship among the Indian and Chinese popula- and Fu’s Fs tests for understanding demographic his- tions (Fig. 3). Broadly, all the H used in this study could tory in Indian and other Tibetan antelope populations be grouped into four clades. However, each clade except (Table 1). Tajima’s D and Fu’s Fs statistics values were the clade 4 in the phylogenetic tree was poorly supported negative, though they were non-significant in the Indian (<0.5 posterior probability), this may have been because population. Similarly, negatives values of Tajima’s D and of haplotype are shared between sampling locations on Fu’s Fs were also reported in earlier studies on Tibetan QTP so do not represent isolated populations. Zhang antelopes [10, 19, 25]. Overall mismatch distribu- et al. [19] also reported low posterior probability for the tion pattern analysis (Fig. 2) of the Indian population majority of the clades except one having high posterior showed weak multimodal; however, shape of patterns probability. may be considered unimodal which suggests signs of In addition, our median-joining (MJ) network (Fig. 4) analysis shows a clear pattern of haplotype distribution which spanned by a total of 46 H with two core hap- Table 2 Estimates of sequence divergence over the lotype (H4 and H42) and appears in star-like topology sequence pairs between groups (lower diagonal) and pair may corroborates a possible population expansion or wise population FST between groups (upper diagonal) the effect of ‘founder effect’. Haplotype 4 seems to be Population IND XJ XZ QH BH ancestral haplotype for all populations, whereas H42 was for the Indian population with a high frequency of IND 0.56222 0.60116 0.57918 0.53813 samples covered. The Indian population is separated XJ 0.019 0.00577 0.0764 0.01928 by one median vector from other Chinese populations, XZ 0.023 0.014 0.03341 0.01196 which is likely to be interpreted as missing haplotype QH 0.028 0.019 0.019 0.00069 that could have remained unsampled for the present BH 0.023 0.015 0.016 0.019 study. Ahmad et al. BMC Res Notes (2016) 9:477 Page 6 of 10

Fig. 2 Bayesian skyline plot for Tibetan antelope of Indian population based on 259 bp control region gene (A) and mismatch distribution graph (B)

Demographic history analysis and star-like topology reveals that the Tibetan antelope population of Xinjiang in the median-joining network analysis undertaken in might have migrated over Kunlun Mountains to the Aksi the present study and other studies [19, 25] have clearly Chin and Ladakh, India. In addition, the presence of H25 revealed historical expansion of the population over (Xinjiang) and H28 (Zhuolaihu Lake) H with major clade its range [25]; however, such analysis has rejected the of Indian populations (Fig. 3) also indicate the presence hypothesis of Ruan et al. [10] that expansion in the popu- of small scale sporadic movements until the recent past lation was at the end of last glacial maximum (LGM). over the Kunlun Mountains. Therefore, we believe that Presence of H in different clades as well as topology in there should be gene flow between the Indian and Xin- MJ network analysis clearly suggest the absence of any jiang Tibetan antelope population if there is no barrier geographic structure in the population of QTP [10, 19, present because geographical distance between Xinjiang 25], whereas the observed five H in the Indian popula- and India is less than that between Xizang and Qinghai tion was not reported in previously studied populations. where large scale migration has been reported [10]. How- The distribution of mixed H among different popula- ever, Sarkar et al. [11] has reported the seasonal migra- tions of QTP (Fig. 3) clearly reveals the presence of high tion in the Indian population during the early period of gene flow among these populations as observed by other snowfall when migrate from Chang Chenmo authors [10, 19, 25]. The observed large scale migrations Valley, India to the Tibet Autonomous Region, of China. by females may have been the reason for the absence of More detailed studies are needed to document the unique haplotype in the four Chinese populations (XJ, extent of movement between these two areas. Arjin Shan XZ, QH, and BH) and may also indicate that no physi- National Nature Reserve (ASNNR) is the largest reserve cal barriers are present along their migratory route on in China and has a high density of Tibetan antelopes in QTP. The absence of H of the western most population the Kunlun Mountains. However, the migration of this in studied populations of QTP so far indicate the lack population in the Xinjiang province was observed mainly of such long range movement, and this may be due to towards the south and to some extent on the Aksai Chin either the presence of any barrier (physical or anthropo- plains, close to Xizang [5, 20]. The Kunlun Mountains are genic) or other ecological reasons. Hence, this requires a one of the longest mountain ranges in Asia, extending detailed study to be undertaken for better understanding more than 2500 km [50] and are known to support per- of environmental and ecological reasoning for such lack manent and migratory populations of Tibetan antelopes of movement between western most and QTP popula- and other [50, 51]. This could have been one tions using other genetic markers or GPS collars. How- of the reasons for the presence of the Xinjiang haplotype ever, major clade II of the Indian population has H of with the clade of the Indian population. However, the Xizang, Zhuolaihu Lake, and Xinjiang. Interestingly, the presence of H10 haplotype of Xizang with the clade of haplotype of Xinjiang (H25) is basal to Indian population the Indian population may indicate the presence of other Ahmad et al. BMC Res Notes (2016) 9:477 Page 7 of 10

Fig. 3 Bayesian phylogenetic relationships among Indian and Chinese populations of Tibetan antelope. All nodes with posterior probability >0.5 are displayed and sequence of Capra hircus (AF533441) was used as outgroup

populations between Indian and Xizang. This is because Conclusions individual females and their calves are known to migrate Analysis of the mtDNA control region (259 bp) indicates to other wintering habitats instead of their original habi- the presence of relatively low genetic diversity in the tats [20]. surviving Tibetan antelope population in India in com- Schaller [5, 21] has recorded migration of the Tibetan parison to what has been reported for Tibetan antelope antelope population of Xinjiang to the Aksai Chin plain populations of QTP. Five H were observed in the Indian close to Xizang and interchange of individuals between population, and so far, these have not been reported in Xinjiang and Xizang populations near Tuzi Lake adja- previously studied populations on QTP. Bayesian phylo- cent to the Chang Tang Reserve as well as close to HeiShi genetic tree analysis reveals the presence of four clades Beihu bordering Xinjiang. It is clear that there has been and indicates lack of population divergence in majority reported migration and exchange of individuals towards of populations. The absence of Indian H in other popu- the western part in its range, but habitat suitability anal- lations and a separate major clade in topology probably ysis is needed for a better understanding of the reasons indicates the absence of female biased migration from for lack of major exchange of individuals between the India to QTP but requires studies to document the extent western most ( close to DBO in north- of migration present in these populations. The Chinese ern Ladakh and Aksi Chin near Kunlun range) and other populations studied so far have a limited geographic populations. structure, and correspondingly, the estimated gene flow Ahmad et al. BMC Res Notes (2016) 9:477 Page 8 of 10

Fig. 4 Median-joining networks showing genetic relationship among control region haplotypes of Pantholops hodgsonii (n 118). Each circle rep- = resents a haplotype and its size is proportional to its total number of individual sequences. Gray dots indicate median vectors; circle colour denotes sampling location of different population

among the four meta-populations (XJ, XZ, QH, and Authors’ contributions MR did DNA extraction and sample collection; VPK carried out DNA extraction, BH) indicates strong historical connections between the PCR assays, DNA sequencing, data analysis, wrote the manuscript; BDJ carried populations and making QTP as panmictic population. out the data analysis, wrote the manuscript and performed demographic, sta- For effective conservation of the western-most popula- tistical and evolutionary analysis; SPG provided the lab facility, conceived the idea, participated in its design, and finalized the manuscript; PN coordinated tion in India, our priority should be to plan genetic stud- in writing the manuscript, assisted in improving the manuscript with their ies to include patterns of genetic diversity, relatedness, comments; KA provided the samples; AAK involved in sample collection. All and population connectivity using mtDNA and nuclear authors read and approved the final manuscript. markers of the reported populations around Ladakh and Author details Aksai Chin to determine their relationship with other 1 Centre for Mountain Wildlife Sciences, Faculty of Veterinary Sciences populations of QTP and identify if any barriers exist. This and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology, Srinagar, Jammu and Kashmir 190006, India. 2 Wildlife would enable us to understand and plan for effective con- Forensic and Conservation Genetics Cell, Wildlife Institute of India, Post Box servation of Tibetan antelopes in the meta-population #18, Chandrabani, Dehradun, Uttarakhand 248001, India. 3 Wildlife Health framework and plan adaptive management for restoring Managment, Wildlife Institute of India, Post Box #18, Chandrabani, Dehradun, Uttarakhand 248001, India. the genetic diversity of the eastern most populations. Besides, Tibetan antelopes will also require strong legis- Acknowledgements lation because continued poaching for high wool demand The authors are grateful to the Director, Dean and Research Coordinator, Wild- life Institute of India, Dehradun, for their strong support and facilitation. Our may influence genetic diversity by reducing effective pop- sincere thanks are due to the Department of Wildlife Protection, Jammu and ulation size as well as population demography. Kashmir, Chief Wildlife Warden, officials and field staff for providing the help and cooperation necessary for conducting the study. The authors acknowl- edge the support provided by the Nodal Officer and all the researchers and Abbreviations staff of the Wildlife Forensic and Conservation Genetics Cell of the Wildlife AKS: Aksai Chin; AAK: Anzara Anjum Khan; ASNNR: Arjin Shan national nature Institute of India for conducting this study. reserve; BH: breeding habitat; CCV: Chang Chenmo Valley; CITES: Convention On International Trade In Endangered Species; DBO: ; DNA: Competing interests deoxyribonucleic acid; IND: India; KA: Khursheed Ahmad; mtDNA: mitochon- The authors declare that they have no competing interests. drial DNA; MR: Mohamed Raza; PN: Parag Nigam; QH: Qinghai; QTP: Qinghai Tibetan Plateau; SPG: Surendra Prakash Goyal; VPK: Ved Prakash Kumar; XJ: Availability of data and material Xinjiang; XZ: Xizang. Generated data submitted in NCBI GenBank, but accession numbers not yet received. Ahmad et al. BMC Res Notes (2016) 9:477 Page 9 of 10

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